With the availability of a burgeoning sequence database, genomic applications demand faster and more efficient methods for the global screening of protein expression in cells. However, the complexity of the cellular proteome expands substantially if protein post-translational modifications are also taken into account.
Dynamic post-translational modification of proteins is important for maintaining and regulating protein structure and function. Among the several hundred different types of post-translational modifications characterized to date, protein phosphorylation plays a prominent role. Enzyme-catalyzed phosphorylation and dephosphorylation of proteins is a key regulatory event in the living cell. Complex biological processes such as cell cycle, cell growth, cell differentiation, and metabolism are orchestrated and tightly controlled by reversible phosphorylation events that modulate protein activity, stability, interaction and localization. Perturbations in phosphorylation states of proteins, e.g. by mutations that generate constitutively active or inactive protein kinases and phosphatases, play a prominent role in oncogenesis. Comprehensive analysis and identification of phosphoproteins combined with exact localization of phosphorylation sites in those proteins (‘phosphoproteomics’) is a prerequisite for understanding complex biological systems and the molecular features leading to disease.
Protein phosphorylation represents one of the most prevalent mechanisms for covalent modification. It is estimated that one third of all proteins present in a mammalian cell are phosphorylated and that kinases, enzymes responsible for that phosphorylation, constitute about 1-3% of the expressed genome. Organisms use reversible phosphorylation of proteins to control many cellular processes including signal transduction, gene expression, the cell cycle, cytoskeletal regulation and apoptosis. A phosphate group can modify serine, threonine, tyrosine, histidine, arginine, lysine, cysteine, glutamic acid and aspartic acid residues. However, the phosphorylation of hydroxyl groups at serine (90%), threonine (10%), or tyrosine (0.05%) residues are the most prevalent, and are involved among other processes in metabolism, cell division, cell growth, and cell differentiation. Because of the central role of phosphorylation in the regulation of life, much effort has been focused on the development of methods for characterizing protein phosphorylation. Many of these phosphorylation sites regulate critical biological processes and may prove to be important diagnostic or therapeutic targets for molecular medicine. For example, of the more than 100 dominant oncogenes identified to date, 46 are protein kinases.
Many cancers are characterized by disruptions in cellular signaling pathways that lead to uncontrolled growth and proliferation of cancerous cells. Receptor tyrosine kinases (RTKs) play a pivotal role in these signaling pathways, transmitting extracellular molecular signals into the cytoplasm and/or nucleus of a cell. Cells of virtually all tissue types express transmembrane receptor molecules with intrinsic tyrosine kinase activity through which various growth and differentiation factors mediate a range of biological effects (reviewed in Aaronson, Science 254: 1146-52 (1991).
The catalytic activity of tyrosine kinases is frequently stimulated by autophosphorylation within a region of the kinase domain termed the activation segment (Weinmaster et al. (1984) Cell 37, 559-568), and indeed this has been viewed as the principal mechanism through which RTKs are activated (Hubbard and Till (2000) Annu. Rev. Biochem. 69, 373-398 and Hubbard, (1997) EMBO J. 16, 5572-5581). Structural analysis of the isolated kinase domains of several receptors has revealed how the activation segment represses kinase activity, and the means by which phosphorylation releases this autoinhibition. In the case of the inactive insulin receptor, Tyr 1162 in the activation segment protrudes into the active site, and the activation segment blocks access to the ATP-binding site (Hubbard et al., (1994) Nature 372, 746-754). Autophosphorylation of Tyr 1162 and two adjacent tyrosine residues repositions the activation segment, thereby freeing the active site to engage exogenous substrates and reorganizing the residues required for catalysis into a functional conformation (Hubbard (1997) EMBO J. 16, 5572-5581). In contrast, the activation segment of the fibroblast growth factor (FGF) receptor is relatively mobile and the tyrosines, which become phosphorylated upon receptor activation, do not occupy the active site. However, the C-terminal end of the FGFR1 activation segment appears to block access to substrate (Mohammadi et al. (1996) Cell 86, 577-587).
Receptor tyrosine kinases within the scope of the present invention include but are not limited to epidermal growth factor receptor (EGFR), PDGF receptor, insulin receptor tyrosine kinase (IRK), Met receptor tyrosine kinase, fibroblast growth factor (FGF) receptor, insulin receptor, insulin growth factor (IGF-1) receptor, TrkA receptor, TIE-1, Tek/Tie2, Flt-1, Flk, VEGFR3, EGFR (HER-1, ERBB2 (HER-2), ERBB3 (HER-3), ERBB4 (HER-4), Ret, Kit, Alk, AxI1, FGFR1, FGFR2, FGFR3 and Eph receptors.
Biological relationships between various human malignancies and disruptions in growth factor-RTK signal pathways are known to exist. For example, overexpression of EGFR-family receptors is frequently observed in a variety of aggressive human epithelial carcinomas, such as those of the breast, bladder, lung and stomach (see, e.g., Neal et al., Lancet 1: 366-68 (1985); Sainsbury et al., Lancet 1: 1398-1402 (1987)). Similarly, overexpression of HER2 has also been correlated with other human carcinomas, including carcinoma of the stomach, endometrium, salivary gland, bladder, and lung (see, e.g. Yokota et al., Lancet 1: 765-67 (1986); Fukushigi et al., Mol. Cell. Biol. 6: 955-58 (1986)). Phosphorylation of such RTKs activates their cytoplasmic domain kinase function, which in turns activates downstream signaling molecules. RTKs are often phosphorylated at multiple different sites, such as distinct tyrosine residues. These enzymes are gaining popularity as potential drug targets for the treatment of cancer. For example, Iressa™, an inhibitor of EGFR, has recently entered clinical trials for the treatment of breast cancer. Similarly, Gleevec™, an inhibitor of BCR/ABL, is now widely used for the treatment of CML. The great advantage of targeted therapeutics, which seek to alter the activity of a single protein, over conventional chemotoxic or radiation therapies is, that they specifically target the deregulated cell and therefore, should not have the wide cytotoxicity and adverse side effects seen with current therapies. Abnormal proliferation, differentiation, and/or dysfunction of cells are considered to be the cause of many diseases. Protein kinases and related molecules play an important role in controlling these cells so that they are very important drug targets.
Protein kinases are critical components of cellular signaling cascades that control cell proliferation and other responses to external stimuli. Modulating these signaling cascades through the inhibition of kinases has the potential to impact many diseases and conditions, including cancer, inflammation, diabetes, and stroke.
Cancer is the second leading cause of death in the western world. Despite advances in diagnosis and treatment, overall survival of patients remains poor. Scientific advances in recent years have enhanced our understanding of the biology of cancer. Human protein tyrosine kinases (PTKS) play a central role in human carcinogenesis and have emerged as the promising new targets. Several approaches to inhibit tyrosine kinase have been developed. These agents have shown impressive anticancer effects in preclinical studies and are emerging as promising agents in the clinic. The remarkable success of BCR-ABL tyrosine kinase inhibitor imatinib (Gleevec™) in the treatment of chronic myeloid leukaemia has particularly stimulated intense research in this field. At least 30 inhibitors are in various stages of clinical development in cancer, and about 120 clinical trials are ongoing worldwide. Innovative approaches are needed to fully evaluate the potential of these agents, and a concerted international effort will hopefully help to integrate these inhibitors in cancer therapy in the near future.
As a result, protein kinases have become one of the most prominent target families for drug development. Hence, there is an urgent need to develop newer more effective therapies to improve patient outcomes.
Rapid scientific advances in recent years have enhanced our understanding of the biology of cancer. Consequently, several novel targets have been identified. Tyrosine kinases have emerged as a new promising target for cancer therapy. Many small molecule kinase inhibitors are currently in development, and the approvals of Gleevec™ (Novartis; leukemia, gastrointestinal tumors) and Iressa™ (AstraZeneca; lung cancer) have validated the inhibition of kinases as a highly promising therapeutic strategy.
Human genome sequence analysis has identified about 518 human protein kinases (constituting about 1.7% of all the human genes). Within this large protein kinase complement, at least 90 tyrosine kinase genes have been identified (58 receptor tyrosine kinases (RTKS, Table 1) and 32 nonreceptor tyrosine kinases (NRTKS, Table 2). The cell signalling pathways they initiate are complex (Schlessinger J. et al. Cell 103 (2000), pp. 211-225). In brief, receptor tyrosine kinases (RTKs) contain an amino-terminal extracellular ligand-binding domain (usually glycosylated), a hydrophobic transmembrane helix, and a cytoplasmic domain, which contains a conserved protein tyrosine kinase core and additional regulatory sequences (that contain crucial C-terminal tyrosine residues and receptor regulatory motifs). Ligand binding (HGF, IGF, EGF, TGF-, or others) to the extracellular domain (ECD) results in receptor dimerisation/oligomerisation, leading to activation of cytoplasmic tyrosine kinase activity and phosphorylation of tyrosine residues (Schlessinger et al., Neuron (1992) 9:383-391). Autophosphorylated tyrosine residues serve as a platform for the recognition and recruitment of a specific set of signal-transducing proteins (such as proteins containing SH2 (Src homology 2) and PTB (phosphotyrosine binding) domains) that modulate diverse cell signalling responses. Nonreceptor tyrosine kinases have a common conserved catalytic domain (similar to RTKs) with a modular N-terminal, which has different adapter protein motifs. Tyrosine kinases play a critical role in the regulation of fundamental cellular processes including cell development, differentiation, proliferation, survival, growth, apoptosis, cell shape, adhesion, migration, cell cycle control, T-cell and B-cell activation, angiogenesis, responses to extracellular stimuli, neurotransmitter signalling, platelet activation, transcription, and glucose uptake (Hunter T. Philos. Trans. R. Soc. Lond., B Biol. Sci. 353 (1998), pp. 583-605). Given their pivotal role in normal homeostasis, it is perhaps not surprising that they have been implicated in several human disorders including developmental anomalies (craniosynostosis syndromes and others), immunodeficiency (severe combined immunodeficiency disease (SCID), hereditary agammaglobulinaemia), non-insulin-dependent diabetes mellitus (NIDDM), atherosclerosis, psoriasis, renal disease, neurological disorders, leukaemia, and solid tumors (Madhusudan S, and Ganesan T S. Clin Biochem. 2004 July; 37(7):618-35).
TABLE 1Receptor tyrosine kinases and cancerTyrosine kinaseCancer associationsEGFR familyEGFR (HER-1)Breast, ovary, lung, glioblastomamultiforme, and othersERBB2 (HER-2)Breast, ovary, stomach, lung,Colon, and othersERBB3 (HER-3)BreastERBB4 (HER-4)Breast, granulosa cell tumorsInsulin R familyIGF-1RCervix, kidney (clear cell),sarcomas, and othersIRR, INSR—PDGFR familyPDGFR-aGlioma, glioblastoma, ovaryPDGFR-βChronic myelomonocyticleukaemia (CMML), gliomaCSF-1RCMML, malignant histiocytosis,glioma, endometriumKIT/SCFRGIST, AML, myelodysplasia,mastocytosis, seminoma, lungFLK2/FLT3Acute myeloid leukaemia (AML)VEGFR familyVEGFR1Tumor angiogenesisVEGFR2Tumor angiogenesisVEGFR3Tumor angiogenesis, Kaposisarcoma, haemangiosarcomaFGFR familyFGFR-1AML, lymphoma, several solidtumorsFGFR-2Stomach, breast, prostateFGFR-3Multiple myelomaFGFR-4—KLG/CCK family (CCK4)—NGFR familyTRKAPapillary thyroid cancer,neuroblastomaTRKBTRKCCongenital fibrosarcoma, acutemyeloid leukaemiaHGFR familyMETPapillary thyroid,rhabdomyosarcoma, liver, kidneyRONColon, liverEPHR familyEPHA2MelanomaEPHA1, 3, 4, 5, 6, 7, and 8—EPHB2Stomach, oesophagus, colonEPHB4BreastEPHB1, 3, 5, and 6—AXL familyAXLAMLMER, TYRO3—TIE familyTIEStomach, capillaryhaemagioblastomaTEKTumor angiogenesisRYK family (RYK)Ovarian cancerDDR family (DDR1Breast, ovarian cancerand DDR2)RET family (RET)Thyroid (papillary andmedullary), multiple endocrineneoplasiaROS family (ROS)Glioblastoma, astrocytomaLTK familyALKnon-Hodgkin lymphomaLTK—ROR family (ROR1—and ROR2)MUSK family (MUSK)—LMR family (AATYK,—AATYK 2, and 3)RTK106—
TABLE 2Nonreceptor tyrosine kinases and cancerTyrosine kinaseCancer associationsABL familyABL1Chronic myeloid leukaemia (CML),AML, ALL, CMMLARGAMLFRK familyBRKBreastFRK—SRMS—JAK familyJAK1LeukaemiasJAK2AML, ALL, T-cell childhood ALL,atypical CMLJAK3Leukaemia, B-cell malignanciesJAK4—SRC-A familyFGRAML, CLL, EBV-associated lymphomaFYN—SRCcolon, breast, pancreas, neuroblastomaYES1colon, melanomaSRC-B familyBLK—HCK—LCKT-cell ALL, CLLLYN—SYK familySYKBreastZAP70—FAK familyFAKadhesion, invasion and metastasis ofseveral tumorsPYK2adhesion, invasion and metastasis ofseveral tumorsACK familyACK1—TNK1—CSK familyCSK—MATK—FES familyFER—FES—TEC familyBMX—BTK—ITK—TEC—TXK—
Tyrosine kinases play a central role in oncogenic transformation of cells. This is achieved in several ways (Blume-Jensen P. et al. Nature 411 (2001), pp. 355-365). Gene amplification and/or overexpression of PTKs (e.g., EGFR and HER-2 overexpression that is commonly seen in several cancers) cause enhanced tyrosine kinase activity with quantitatively and qualitatively altered downstream signalling. Genomic rearrangements (like chromosomal translocation) can result in fusion proteins with constitutively active kinase activity (e.g., p210BCR-ABL fusion protein seen in chronic myeloid leukaemia). Gain of function (GOF) mutations or deletion in PTKs within the kinase domain or extracellular domain result in constitutively active tyrosine kinase (e.g., EGFRvll mutant that lacks amino acids 6-273 of the extracellular domain is constitutively active and is seen in solid tumors). Autocrine-paracrine stimulation by overexpression of ligands results in persistent tyrosine kinase stimulation (e.g., TGF- is overexpressed in glioblastoma and head and neck cancer (Grandis J. R. et al. J. Cell. Biochem. 69 (1998), pp. 55-62). Finally, retroviral transduction of a protooncogene corresponding to a PTK concomitant with deregulating structural changes is a frequent mechanism by which oncogenic transformation occurs in animals (rodents and chicken) (Blume-Jensen P. et al. Nature 411 (2001), pp. 355-365).
A significant number of tyrosine kinases (both receptor and nonreceptor types) are associated with cancers. Clinical studies suggest that overexpression/deregulation of tyrosine kinases may be of prognostic/predictive value in patients (i.e., may indicate an aggressive tumor biology or may predict poor response to therapy and shorter survival). EGFR family of tyrosine kinases is the most widely investigated. EGFR (HER-1) overexpression is associated with a poor prognosis in ovarian, head and neck, oesophageal, cervical, bladder, breast, colorectal, gastric, and endometrial cancer (Nicholson R. I et al. Eur. J. Cancer 37 Suppl. 4 (2001), pp. S9-S15). HER-2 overexpression is associated with poorer outcome in patients with breast (Tandon A. K. et al. A. K. Clin. Oncol. 7 (1989), pp. 1120-1128), ovary Meden H. et al. Eur. J. Obstet. Gynecol. Reprod. Biol. 71 (1997), pp. 173-179), prostate (Sadasivan R. et al. J. Urol. 150 (1993), pp. 126-131), lung (Selvaggi G. et al. Cancer 94 (2002), pp. 2669-2674) and bone cancer (Zhou H. et al. J. Pediatr. Hematol. Oncol. 25 (2003), pp. 27-32). Mutation in C-KIT tyrosine kinase is associated with inferior survival in patients with gastrointestinal stromal tumors (Taniguchi M. et al. Cancer Res. 59 (1999), pp. 4297-43) and adversely affects relapse rate in acute myeloid leukaemia (Care R. S. et al. Br. J. Haematol. 121 (2003), pp. 775-777). In small cell lung cancer, C-KIT expression was linked to poor survival (Naeem M. et al. Hum. Pathol. 33 (2002), pp. 1182-1187). The expression of IGF-1R along with IGF-1 and IGF-2 may have prognostic value in a subset of colorectal cancer patients (Peters G. et al. Virchows Arch. (2003). In acute myeloid leukaemia, FLT 3 mutation predicts higher relapse rate and a shorter event free survival (Schnittger S. et al. Blood 100 (2002), pp. 59-66). VEGF is a central growth factor that drives tumor angiogenesis and is an important prognostic marker in solid tumors (Fox S. B. et al. Lancet Oncol. 2 (2001), pp. 278-289). Recent studies suggest that VEGFR 3 expression in lung cancer is associated with a significantly lower survival rate (Arinaga M. et al. Cancer 97 (2003), pp. 457-464) and in colorectal cancer, it may have prognostic significance (Parr C. et al. Int. J. Oncol. 23 (2003), pp. 533-539). Trk tyrosine kinase is an important marker for neuroblastoma (NB). TrkA is present in NB with favourable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavourable, aggressive NB with MYCN-amplification (Eggert A. et al. Klin. Padiatr. 212 (2000), pp. 200-205). HGFR (Met) overexpression is associated with disease progression, recurrence, and inferior survival in early-stage invasive cervical cancer (Baycal C. et al. Gynecol. Oncol. 88 (2003), pp. 123-129) correlates with poor prognosis in synovial sarcoma (Oda Y. et al. Hum. Pathol. 31 (2000), pp. 185-192) and predicts a significantly shorter 5-year survival in hepatocellular carcinoma (Ueki T. et al. Hepatology 25 (1997), pp. 862-866). Axl tyrosine kinase expression was associated with poor outcome in acute myeloid leukaemia (Rochlitz C. et al. Leukemia 13 (1999), pp. 1352-1358). Tie-1 kinase expression inversely correlates with survival in gastric cancer (Lin W. C. et al. Clin. Cancer Res. 5 (1999), pp. 1745-1751) and in early chronic phase chronic myeloid leukaemia (Verstovsek S. et al. Cancer 94 (2002), pp. 1517-1521). Soluble Tie-2 receptor levels independently predict loco-regional recurrence in head and neck squamous cell (Horner J. J. et al. Head Neck 24 (2002), pp. 773-778). ALK protein expression is an independent predictor of survival and serves as a useful biologic marker of a specific disease entity within the spectrum of anaplastic large cell lymphoma (ALCL, Gascoyne R. D. et al. Blood 93 (1999), pp. 3913-3921). Src tyrosine kinase is an independent indicator of poor clinical prognosis in all stages of human colon carcinoma (Aligayer H. et al. Cancer 94 (2002), pp. 344-351). BCR-ABL tyrosine kinase is of prognostic value and predicts response to therapy in haematological malignancies including chronic myeloid leukaemia (Olavarria E. et al. Blood 97 (2001), pp. 1560-1565 and O'Dwyer M., et al. Oncologist 7 Suppl. 1 (2002), pp. 30-38) and acute lymphoblastic leukaemia (Gleissner B. et al. Blood 99 (2002), pp. 1536-1543) FAK overexpression is correlated with tumor invasiveness and lymph node metastasis in oesophageal squamous cell carcinoma (Miyazaki, T. et al. Br. J. Cancer 89 (2003), pp. 140-145) and reduced expression of the Syk gene is correlated with poor prognosis in breast cancer (Toyama T. et al. Cancer Lett. 189 (2003), pp. 97-102).
Several approaches to target tyrosine kinases have been developed. Tyrosine kinase domain inhibitors, tyrosine kinase receptor blockers (e.g., monoclonal antibodies), ligand modulators (e.g., monoclonal antibodies), RNA interference and antisense technology, gene therapy strategy, inhibitors of Src tyrosine kinase, BCR-ABL inhibitors, downstream signal transduction pathway inhibitor are potential strategies for cancer therapy. Classification of such inhibitors based on their mode of action is summarized in Table 3. Receptor tyrosine kinases are multidomain proteins. The catalytic domain (Mg-ATP complex binding site) has emerged as the most promising target for drug design in recent years. Random screening of compound libraries initially identified small molecule chemical inhibitors of the catalytic domain. Combinatorial chemistry, in-silico cloning, structure-based drug design, and computational chemistry have now become indispensable tools in lead compound identification and optimisation of these inhibitors. Highly sensitive, accurate, and reliable high throughput assays for screening inhibitors have been developed (including scintillation proximity assay, fluorescence polarisation assay, homogenous time-resolved fluorescence assay, and the heterogeneous time-resolved dissociation-enhanced fluorescence technology (F. A. Al-Obeidi and K. S. Lam, Oncogene 19 (2000), pp. 5690-5701). Knowledge about tertiary structure of protein kinases has expanded, and the X-ray crystallographic structure for over 50 protein kinases has been resolved. Understanding of the molecular interactions of the various parts of the ‘ATP-binding site’ (adenine region, sugar region, hydrophobic pocket, hydrophobic channel, and the phosphate-binding region) has accelerated drug development (Fabbro D. et al. Pharmacol. Ther. 93 (2002), pp. 79-98).
TABLE 3Classification of inhibitorsSmall molecule inhibitorsLigand modulationTargeting EGFRTargeting VEGFZD1839 (Iressa, Gefitinib)Bevacizumanb (RhuMAb, Avastink)OSI-774 (Tarceva, Erlotinib,MV833CP-358774)Soluble Flt-1 and Flk-1PKI-166VEGF TrapCI-1033 (PD183805)GFB 116CGP-59326ANM3EKB-569VEGF 121-diphtheria toxinGW 572016conjugateTargeting HER-2/neuTargeting EGFPKI-166 (also inhibitsDAB389EGF (diphtheria toxinEGFR)conjugate)TAK165Targeting FGFGE-572016 (inhibits EGFR)Interferon-a (reduces FGFCI-1033 (pan erbBproduction)inhibitor)Targeting VEGFRMonoclonal antibodies againstSU5416 (also targets FLT3)receptorsZD4190Targeting EGFRPTK787/ZK222584IMC-C225 (Cetuximab)CGP 41251ABX-EGFCEP-5214Y10ZD6474 (also inhibits RET)MDX-447 (EMD 82633)BIBF1000h-R3VGA1102EMD 72000SU6668 (also inhibitsTargeting HER-2/neuPDGFR and FGFR)Herceptin (trastuzumab)Targeting PDGFRMDX-H210SU11248 (also inhibits2C4 (pertuzumab)C-KIT, FLT-3)Targeting VEGFRCGP-57148IMC-1C11 (anti-KDR antibody)Tricyclic quinoxalinesAnti-Flt-1 antibody (MF1)(also targets C-KIT)Targeting FGFRGene therapy approachesSU4984Targeting EGFRSU5406Antisense oligonucleotideTargeting BCR-ABLTargeting VEGF/VEGFRSTI571 (Glivec) (alsoAntisense oligonucleotidestargets C-KIT, PDGFR)Adenovirus-based Flt-1 gene therapyNSC680410Retrovirus-based Flk-1 gene therapyTargeting C-KITRetrovirus-based VHL gene therapyPD166326 (also targetsAngiozymeBCR-ABL)Targeting IGF-1RPD1173952 (also targetsINX-4437 (Antisense oligonucleotides)BCR-ABL)Targeting FLT3OthersCT53518APC8024 (vaccine against HER-2GTP14564overxpressing cells)PKC412AP22408 (Src SH2 domain inhibitor)Targeting SrcB43-genistein conjugatePP1 (also inhibits C-KIT,AG538 (IGF-1R inhibitor)BCR-ABL)PD116285CGP77675CGP76030Targeting TRKCEP-701 (also inhibits Flt 3)CEP2583
Although ATP-binding site is highly conserved among tyrosine kinases, minor differences in kinase domain architecture have allowed development of highly selective inhibitors (Levitzki A. Eur. J. Cancer 38 Suppl. 5 (2002), pp. S11-S18). Data on EGFR co crystallised with its inhibitor OSI-774 (Tarceva™) were published recently and provide valuable insight into the mechanism of action of this compound (Stamos J. at al. J. Biol. Chem. 277 (2002), pp. 46265-46272). Most small molecules in clinical development bind in the vicinity of the ATP-binding site of their target kinases, using a part of their scaffold to mimic the binding of the adenine moiety of ATP. Such ATP mimics are competitive inhibitors of the substrate-binding sites within the catalytic domain (Laird A. D. et al. Expert Opin. Invest. Drugs 12 (2003), pp. 51-64 and Fry D. W. Exp. Cell Res. 284 (2003), pp. 131-139) and compete with endogenous ATP (often present in millimolar levels in cells) for binding. Early potent lead compounds had poor solubility and required extended multiple dosing schedules to achieve and maintain adequate plasma levels in patients necessary for optimal target inhibition. To increase solubility, new compounds were generated, but they had reduced affinity to the kinase domain. To circumvent these problems, irreversible inhibitors are now being developed in the hope that covalent attachment of a selective inhibitor to the kinase domain would completely abolish catalytic activity and would translate into potent drugs (Denny W. A. et al. Pharmacol. Ther. 93 (2002), pp. 253-261). Two such inhibitors are in advanced stage of development (CI-1033) (Pfizer) and EKB-569 (Wyeth) that bind irreversibly to EGFR and HER-2, respectively (Laird A. D. et al. Expert Opin. Invest. Drugs 12 (2003), pp. 51-64). Small molecules that target more than one tyrosine kinase have also been developed, and they have the potential to block multiple pathways and produce enhanced anticancer effect (Table 3). PKI-166 inhibits EGFR and HER-2 (Mellinghoff I. K. et al. Cancer Res. 62 (2002), pp. 5254-5259CI-1033) is a pan ErbB inhibitor (Slichenmyer, W. J. et al. Semin. Oncol. 28 (2001), pp. 80-85), SU6668 inhibits VEGFR, PDGFR, and FGFR (Hoekman K. et al. 7 Cancer J. Suppl. 3 (2001), pp. S134-S13, and STI 571 inhibits BCR-ABL, C-KIT, PDGFR, and ARG (Buchdunger, E. et al. Eur. J. Cancer 38 Suppl. 5 (2002), pp. S28-S36. and Nishimura N. et al. Oncogene 22 (2003), pp. 4074-4082.
In the 1980s, first natural tyrosine kinase inhibitors quercetin and genistein were reported (Akiyama T. et al. J. Biol. Chem. 262 (1987), pp. 5592-5595 and J. Mendelsohn J. J. Clin. Oncol. 20 (2002), pp. 1S-13S). Since then, an overwhelming number of natural and synthetic small molecules inhibitors have been described. Tyrosine kinase inhibitors can be broadly categorised into natural products and related derivatives (quercetin, genistein, staurosporine, erbastatins, clavilactones); quinazolines, pyridopyrimidines, and related heterocyles (e.g., ZD1839); phenylamino-pyrimidines (e.g., STI 571); tryphostins and analogues (e.g., SU1498, SU101, SU0020); indoles and oxindoles (e.g., SU5416, SU6668, SU5402; F. A. Al-Obeidi and K. S. Lam, Oncogene 19 (2000), pp. 5690-5701).
One of the major difficulties in the development of small molecule kinase inhibitors is specificity (McMahon et al. (1998) Curr. Op. in Drug Discovery and Dev. 1(2), 131-146). Most compounds currently target the highly conserved ATP binding site of kinases, and therefore tend to bind and inhibit more than one enzyme in the class. Because there are more than 500 human protein kinases (Manning et al., Science (2002) 298, 1912) and inhibition of multiple kinases (or the “wrong” kinase) may lead to adverse effects, it is critical to assess compound specificity. However, the problem has been that most “off-target” interactions are not predictable and the development of conventional experimental activity assays for kinases is very time consuming and resource intensive. As a result, even though compound specificity is critically important to assess, it has been extremely difficult, if not impossible, to do so comprehensively and systematically. Protein kinases are key regulators of most cellular signaling pathways in eukaryotic cells. Many protein kinase inhibitors have been developed to study specific functions of kinases in signaling pathways and as potential therapeutic agents (Cohen, P. (2002) Nat. Rev. Drug Discov. 1, 309-315) Because of the large size of the protein kinase superfamily (>500 human) and the fact that most kinase inhibitors bind in the highly conserved ATP-binding pocket, it is widely accepted that kinase inhibitors inhibit more than one target (Davies, S. P., Reddy, H., Caivano, M. & Cohen, P. (2000) Biochem. J. 351, 95-105). As a result, the inhibitors used as chemical tools to probe the often poorly understood roles of kinases in signaling pathways are paradoxically of incompletely characterized specificity. The same is true for kinase activators. The present invention is also usable for the parallel profiling of kinase activators of multiple kinases in one cavity.